Method for the determination of biochemical data

ABSTRACT

Microorganisms and unicellular organisms in a sample are identified or determined by exposing the sample to an adsorbent having a specific binding power for the entity to be determined to bind the entity to the adsorbent, separating unbound sample, exposing the adsorbent containing the entity to a nutrient medium to initiate metabolism with resulting change in the physical or chemical characteristics of the substrate and observing these changes.

The present invention concerns a method for the determination of thecontent and species of microorganisms in a sample. There are alreadyseveral different procedures to determine species and amount, but acommon drawback with all these is that they require a long time, oftenseveral days to make one determination. To date workers have used aprocedure involving plating, which means spreading, on a plate or glasssurface, a layer of substrate to which is added the microorganisms whosenumber and species. At first nothing is seen in the medium, but laterare to be determined on the microorganisms divide and eventually form anumber of colonies that are observable by the naked eye. Based on theassumption that one cell creates one colony, the number ofmicroorganisms are allowed to divide, the species of the microorganismcan be determined. Another procedure to determine amount and kind ofmicroorganism is to use specific sorbents containing antibodies. Suchsorbents immobilize a predetermined species of microorganism againstwhich the antibodies are directed. When the cells have been captured inthis way they can be incubated in a growth medium, and when themicroorganisms, through division, have given rise to a larger group ofcells that is observable by the naked eye, the amount of microorganismscan be determined by colony counting. The choice of substrate to whichthe adsorbent with the microorganisms are added gives an indication ofthe kind of microorganisms that has been adsorbed, since only certainmicroorganisms are able to grow in a certain substrate, while othersneed a different substrate for their growth.

The present invention is aimed at creating a system of procedures bywhich it is possible to determine biochemical data concerningmicroorganisms within a short period of time, in many cases within twohours.

The procedure is based upon the use of an adsorbent for a preselectedmicroorganism, where the sample from which the microorganism is boundtogether with the sorbent, is washed after the adsorption the in orderto eliminate all non-specifically bound cells. The washed microorganismsbound to the sorbent are exposed to a substrate so that the cellmetabolism is started. For this metabolism there is a need for certainsubstances concomittant with generation of metabolic products.

The quantity of generated products is directly related to the number ofadsorbed cells. One or more products may be utilized either directly orindirectly to determine the amount of microorganisms. The species of themicroorganisms is determined by the adsorbent due to its specificbinding properties.

The adsorbent is as a rule a carrier provided with antibodies directedagainst the microorganisms that is to be adsorbed. If it is desirable toadsorb all microorganisms, then as an alternative either mixtures ofantibodies or other binding structures with a broader specificity, eg.lectins are used.

The above mentioned substrate may be or may contain oxygen and theproducts may be eg. those generated by consumption of oxygen due tometabolic activity. The amount of oxygen consumed can be determined withestablished measuring equipment.

Other parameters may be the pH of the substrate-medium. The pH-value canbe determined by using a pH-indicator and fotometric equipment.

Still another parameter is reduction equivalents formed during themetabolism. These reduction equivalents are uncoupled from the ordinarymetabolism by eg. artificial electron acceptors, the degree of reductionof which can be determined by established technique.

Still another parameter may be the amount of carbon-dioxide generatedduring the metabolism, as this amount can be determined by usingestablished analytical instruments.

Moreover, for certain types of cells specific metabolites may beexcreted, the concentration of which in the medium can be used todetermine the number of cells bound to the sorbent. Analysis in such acase is performed with a dedicated specific analysis developed for theindividual metabolites.

Still another physical property of the medium that is changed during themetabolic events is the conductivity. By measuring same and the changesin conductivity over a certain time of incubation, the quantity ofmetabolizing cells or the degree of metabolic activity in a fixed numberof cells can be determined.

The procedure according to the present invention can furthermore also beused to determine the concentration of certain biochemical products thatin a clear way influences on the metabolism of the microbial cells.

Examples of such substances are vitamins and antiobiotics. Analyses ofsuch substances are carried out using predetermined numbers of aspecific microorganism which is bound to the sorbent.

When assaying the content of a vitamin in a nutrient, thenmicroorganisms are bound to the sorbents that have an absoluterequirement for the actual vitamin for their metabolism. The sample tobe analysed is mixed with a full medium for the cells from which thesubstance to be analysed for has been omitted. Then, to the washedsorbent with adsorbed microorganism the substrate that is to be analysedis added. The degree of metabolic activity is dependent upon the amountof the vitamin present. By measuring metabolic activity for the cellpreparations at known concentrations of vitamins, the metabolicactivities registered can be used as reference values when the contentin an unknown sample is to be evaluated from the calibration curve.

The procedure according to the invention can also be used to determinethe influence of inhibiting substances on cell metabolism, eg.antibiotics. In this case the adsorbent is charged with a certainmicroorganism in a predetermined amount. Antibiotics are introducedtogether with a nutrient medium to a washed adsorbent with adsorbedmicroorganisms. After metabolic activity has occurred for a certainperiod of time, the change in concentration of metabolites can be readeg. as a change in pH-value and thus it is possible also to determinethe amount antibiotics in the sample by comparative studies with acalibration curve which is obtained with known concentrations ofantibiotics.

Additional characteristics of the present invention will appear below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described more in detail below with reference to thedrawing wherein

FIG. 1 shows a calibration curve for a predetermined microorganism,where the product generated, here read as change in absorbance, isplotted versus the number of microorganisms, N;

FIG. 2 shows a calibration curve valid for a predetermined vitamin, withthe concentration of vitamin C plotted against measured change inabsorbance after a given period of incubation of a predetermined numberof cells;

FIG. 3 shows a calibration curve valid for a predetermined antibioticand where the absorbance read is plotted versus concentration ofantibiotic in the sample A;

FIG. 4 shows a simple unit for practically performing the presentinvention.

In the present invention it is essential pro primo, to use an absorbentto which cells are adsorbed, pro secundo to have a washing unit wherethe adsorbent with bound cells is washed to remove all unspecificallybound material and pro tertio to have a unit where adsorbed cells aresupplied with nutrients so that cell metabolism can take place. Thisthree-step system must, in order to be useful, be calibrated and this isdone by constructing calibration curves from the behaviour of knownamounts of known microorganisms. By constructing the curves, one usesthe changes in physical parameters that take place as a result of themetabolism of the cells. These parameters may be acidity (read atpH-value), oxygen tension, redox-level, conductivity, concentration of acertain metabolite, carbon dioxide pressure etc.

One of the most useful parameters is the pH-value. To easily determinethis a pH-electrode may be used, but in most cases a colouredpH-indicator has been used, the colour of which is related to thepH-value in the solution. Such pH-indicators may be of very differentchemical structures. Several different ones have been used in thissystem. Examples of suitable substances are heptametoxired andphenolred. Usually the indicator is added to the nutrient medium and thenutrient medium used is analysed, usually spectrophotometrically. Afterthe above mentioned three step procedure has been calibrated againstknown quantities of the species to be analysed, the analytical systemcan be used to primarily determine amount of a certain microorganism ina sample. The capture of the above mentioned predetermined microorganismis carried out by the use of a carrier-substance to which antibodiesdirected against the microorganism that shall be investigated are bound.As carrier is usually used a gel with a low tendency of unspecificadsorption of cells and other biological material. A frequently usedcarrier in such cases is Sepharose. The carrier must be modified byattacking an antibody to it--an antibody with specificity for themicroorganism that shall be quantified in the analytical procedure. Incertain cases it may also be of interest to collect the whole spectrumof microorganisms, and in that case a sorbent with the ability to adsorbmicroorganisms generally must be used. Such an adsorbent can be based ona binding structure of lectin-type, which is well-known, in many cases,to have a much broader substrate specificity than specific antibodiesusually have.

If in a milieu, the amount of a certain microorganism is to bequantified, then an adsorbent is produced suitable for binding thespecific microorganism. The adsorbent with its binding entities, eitherlectins or antibodies, is allowed to be in contact with the milieu wherethe microorganisms are present. After the absorbent has been exposed tothe microorganisms for a given period of time, the sorbent is washed toremove unspecifically bound cells.

The washed sorbent with microorganisms is then supplied with nutrient,which can be a sugar solution with suitable buffering substances and eg.a pH-indicator, or some other liquid medium, in which the cells to bequantified can metabolize.

When the absorbent with the captured microorganisms is exposed tonutrient medium, metabolism starts and is allowed to proceed for a fixednumber of minutes, usually 30-120. After incubation in the nutrientmedium analysis is performed of the medium, usually by aspectrophotometer. Based on the value read in that analysis, it ispossible, by consulting a calibration curve (FIG. 1), to determine thenumber of microorganisms of the selected species in relation to thenumbers used as references when setting up the calibrated curve for aselected organism. In the case here described there has, been used as aparameter change in pH-value as a result of metabolic activity. Inmetabolism there is in many cases a demand for externally added oxygen.The amount of oxygen consumed can be determined and from this value itis possible to determine the number of cells by using a calibrationcurve similar to that in FIG. 1. In a similar way the number ofredox-equivalents as well as the amount of carbondioxide generated bythe metabolic activity can be determined. These values are directlyrelated to the number of microorganisms.

The way in which the number of microorganisms discussed so far arequantified is generally by using laboratory equipment well-known tochemists. However, it is also possible to use the present invention in amore basic way. One prerequisite however is that a calibration curve ofthe type shown in FIG. 1 be available and that it is possible, by theuse of charts with different colours or possibly by spectrophotometry,to quantify the change in colour that appears in the nutrient medium asa result of metabolism by the investigated microorganisms. Thesimplified method for quantification is illustrated in FIG. 4 with thesubfigures 4a-4f.

In these figures is shown a disposable plastic syringe 5, with a piston6, and a piston handle 7. Just in front of the tip of the syringe isplaced a filter 8, eg. of nylon with holes of 25 μm. On top of thefilter is placed the adsorbent equipped with antibodies or other bindingstructures directed against the microorganism to be quantified. Thesyringe 5 is dipped into a vessel 9, containing a fluid with themicroorganisms to be quantified. The fluid with the microorganisms issucked into the syringe 5, where the adsorbent captures that amount ofmicroorganisms that it is capable of under the conditions used. Then theliquid is removed together with non-bound microorganisms and otherimpurities. This is done according to a washing procedure as illustratedin FIG. 4c. In 4d a nutrient medium is sucked into the syringe and mixedwith the sorbent containing captured microorganisms. The nutrient mediummay consist of a buffered glucose solution with suitableindicator-molecules or other substances.

After a sufficient amount of nutrient medium is drawn into the syringe5, it is removed from the vessel 11. The nutrient medium is kept in thesyringe 5, and is allowed to react for a certain time, usually between30 and 120 minutes, sometimes shorter or longer. After this time periodthe syringe 5 is emptied of nutrient medium. The nutrient medium isadded to a vessel 12 that preferentially is transparent. By measuringthe colour of the nutrient medium the amount of microorganisms in thesample 9 can be determined.

The procedure according to the present invention can also be used todetermine concentration of vitamin in a substrate. Hereby, the procedureinvolves the selection of a microorganism that needs an external supplyof the vitamin of interest in order to be capable of maintainingmetabolism. An adsorbent with such microorganisms is washed in the usualway and is then exposed to a nutrient medium that is a complete mediumwith the exception of the vitamin to be quantified, mixed with thesample to be analysed. The degree of metabolic activity is related tothe content of vitamins in the sample. In this case also changes inpH-value can be utilized for determining metabolic activity and,thereby, via a calibration curve, the vitamin content. To simplify theprocedure a calibration curve must be constructed according to FIG. 2which can be used to evaluate the vitamin content by measured colourintensity.

The assay unit according to the present invention can also be used todetermine the concentration of a known antibiotic of studying theinfluence by various samples containing antibiotic on the metabolism ofvarious microorganisms. In this procedure, the adsorbent is exposed to,and thus can adsorb a predetermined number of microorganisms. Afterwashing of the exposed adsorbent to remove all cells not properly bound,it is treated with a nutrient medium in combination with an antibiotic.Metabolism starts operating, but it will be inhibited due to the factthat the antibiotic to a certain extent inhibits the activity of themicroorganisms. After a certain time of exposure, which usually isbetween 30 and 120 minutes, the colour intensity of the nutrient mediumcan be read and thereby the influence of the antibiotic can beevaluated. In FIG. 3 is shown a calibration curve obtained from using acertain number of cells incubated in media containing varyingconcentrations of the antibiotic to be quantified. Translation of valuesobtained on unknown samples can be done by using a calibration curve. Asa further information from such a test, the pattern of resistance forthe microorganism can be determined.

All cells contain ATP. This fact can be used to determine the number ofcells in a sample. By using the above mentioned adsorption technique thecells are bound to a specific adsorbent. The bound cells are thentreated so that ATP is released to the medium surrounding the sorbent.The amount of released ATP has been shown empirically to be correlatedto the number of cells. By addition of the enzyme-substrate systemluciferas--luciferin a light signal can be obtained that can be relatedto the amount of ATP in the sample and thus to the number of cells inthe primary sample.

This latest described application concerns still another important areaof application of general interest, namely concentration of cells from asample to a small volume in order to obtain a sensitive and accurateanalysis.

EXAMPLE 1 (COUNTING OF YEAST CELLS)

A disposable plastic syringe (2 ml) equipped with a 25 μm-filter infront of the outlet is supplied with 1 ml Concanavalin A-Sephadex. Then1/2 ml of sedimented Concanavalin A Sepharose (either prepared byourselves according to established procedures or bought from PharmaciaFine Chemicals AB, Uppsala, Sweden) is suspended in 2 ml buffer, pH 7.4(0.004 mM KH₂ PO₄, 0.054 mM Tris, 0.41 mM NaHCO₃, 0.95 mM CaCl₂, 0.80 mMMgSO₄, 5.36 mM KCl, 137 mM NaCl) and poured into the syringe. The pistonis then introduced and the air is emptied before the excess of liquid isforced out. The sample to be analysed is sucked into the syringe. Atotal of 2 ml of sample is sucked in. Incubation is carried out for 10minutes to allow the cells to bind to the gel. During incubation thecontent of the syringe is mixed gently by turning the syringeend-over-end with time intervals of 1-3 minutes. After this incubation,the solution is forced out of the syringe and 2 ml of buffer is suckedin. The solution is forced out again. This washing procedure is repeated4 times before 2 ml of substrate is sucked in after which the syringe isshaken so that a homogeneous suspension is obtained. The excess ofsubstrate is emptied so that 0.8 ml (total volume=substrate+gel) remainsin the syringe.

The substrate solution consists of a buffer having the composition givenabove, supplied with glucose to a final concentration of 50 mM andneutral red to a final concentration of 0.2 mg/ml. By the addition ofindicator to the medium pH must be adjusted to the initial value.

Incubation is carried out for 2 hrs in a water bath at 26° C. Duringthis time period mixing is achieved by intermittent shakings of thesyringe.

After the incubation period, the substrate solution was forced out ofthe syringe and collected in a collecting vessel. The absorbance at 528nm was read.

    ______________________________________                                        cell number   change absorbance                                               ______________________________________                                          4-10.sup.4  0.054                                                             7-10.sup.4  0.057                                                           1.1-10.sup.5  0.078                                                           1.5-10.sup.5  0.085                                                           2.2-10.sup.5  0.088                                                           2.5-10.sup.5  0.095                                                           7.5-10.sup.5  0.107                                                           1.0-10.sup.5  0.119                                                           ______________________________________                                    

EXAMPLE 2 Concentration and counting of yeast cells in various dilutedsolutions

In this example a 2 ml disposable plastic syringe was used in which 0.5ml Concanavalin-S-Sepharose had been packed according to the proceduredescribed in the previous example. This small column was used in thisexample of application as an adsorbent through which the solution to beanalysed was drawn with a constant speed. Standard solutions withvarying dilutions of yeast cell suspensions were prepared by diluting ayeast cell suspension with the buffer described in Example 1.

The volume of the sample sucked into the sorbent was adjusted withrespect to the cell number, so that in total an equally large number ofcells were introduced in the Concanavalin A-Sepharose column. When thecell suspension had been drawn through the column, incubation wasallowed to proceed for 10 minutes thereby giving possibilities for thecreation of strong so called multi-point attachment bonds. Theexperimental procedure followed that given in Example 1,

In the experiment, except for the blank experiment, 6 milj cells weresucked into the sorbent.

    ______________________________________                                        No of cells per ml                                                                           Absorbance at 528 nm                                           ______________________________________                                        0              0.020                                                             .sup. 0.1 milj                                                                            0.080                                                          0.2            0.116                                                          0.6            0.115                                                          1.2            0.120                                                          6              0.122                                                          ______________________________________                                    

EXAMPLE 3 Quantification of thiamin by yeast cells

The practical details in essence follow that given in Example 1,however, with another buffer, substrate and indicator. Furthermore, thecondition of the cells in the starting moment is quite different. Theyare grown on Difcos special medium for thiamine assay. (The cells weregrown for two days according to Difcos manual).

Buffer: 0.1M sodium acetate/acetic acid pH 4.5 containing also 0.95 mMCaCl₂, 0.81 mM MgSO₄, 5.36 mM KCl, 137 mM NaCl and 1.0 mM MnCl₂.

Indicator: Bromophenylblue dissolved in the same buffer, 4 mg/l.

Thiamine-solution: After capturing the cells by the ConcanavalinA-Sepharose, the syringe was filled (2 ml/syringe) for 30 minutes with athiamine solution or a sample solution where the content of thiamin wasto be determined. Incubation was carried out at 37° C. with regularmixing. The thiamin solution was afterwards washed out through 2repeated washings with buffer.

To each syringe was then added substrate, 50 mM glucose in buffer withindicator 4 mg/L. Incubation for 2 hrs at 37° C. under gentle mixing.The substrate solution is emptied and A₅₈₅ is read.

    ______________________________________                                        Thiamine μg/ml                                                                              A.sub.585                                                    ______________________________________                                        0                0                                                            0.1               0.0253                                                      0.05             0.040                                                        0.10             0.047                                                        0.50              0.0585                                                      1.0              0.062                                                        ______________________________________                                    

EXAMPLE 4 Quantification of E. coli cell

The basic experimental procedure was the same as given in Example 1.

Buffer used: CaCl₂, 0.95 mM, KCl 5.35 mM, NaCl 137 mM, MgSO₄ 0.80 mM,KH₂ PO₄ 0.004 mM NaHCO₃ 0.04a mM Tris 0.053 mM, pH 7.4.

Indicator: Neutralred 0.2 mg/ml substrate: 50 mM glucose in the abovebuffer.

In a 2 ml disposable syringe was added 0.5 ml Sepharose 4B to which wascoupled antibodies with binding specificites against Hemophilusinfluencae, but also a certain activity against E. coli. The couplingtechnique used is known (C. Borrebaeck, J.Borjesson and B. Mattiasson,Clin. Chim. Acta 86, (1978) 267-278). Incubation for 20 minutes wasused. After washing with buffer incubation with substrate and indicatortook place for 2 hrs at 37° C. Then the absorbance was read at 528 nm.

    ______________________________________                                               Number of cells                                                                         A.sub.528                                                    ______________________________________                                               1 10.sup.6                                                                              0.030                                                               2 10.sup.6                                                                              0.058                                                               3 10.sup.6                                                                              0.080                                                               4 10.sup.6                                                                              0.103                                                               5 10.sup.6                                                                              0.110                                                        ______________________________________                                    

EXAMPLE 5 Quantification of amphotericin

Quantitation of amphotericin is done by measuring the inhibiting effectof amphotericin on metabolic response from immobilized cells.

The procedure follows Example 1, with the exception that 6 ml cells wereused and exposed to varying concentrations of amphotericin for 20 minprior to incubation in substrate.

    ______________________________________                                        Amphotericin μg/ml                                                                            A                                                          ______________________________________                                        0                  0.067                                                      2                  0.055                                                      4                  0.051                                                      8                  0.042                                                      16                 0.038                                                      32                 0.041                                                      ______________________________________                                    

I claim:
 1. A method for the identification or determination of anentity selected from the group consisting of microorganisms andunicellular organisms in a sample which comprises:(a) exposing thesample to an adsorbent having a specific binding power for said entityto bind said entity to said adsorbent; (b) separating unbound samplefrom the adsorbent; (c) exposing the adsorbent with bound entity to anutrient-containing substrate to initiate metabolism of the entity,thereby causing the substrate to change its chemical or physicalcharacteristics; and (d) observing the changes of chemical or physicalcharacteristics of the substrate caused by the metabolic process.
 2. Themethod of claim 1, wherein the adsorbent contains immobilized lectins.3. The method of claim 1, wherein the adsorbent contains immobilizedantibodies.
 4. The method of claim 1, wherein the changes in the pH ofthe substrate resulting from the metabolic process are observed.
 5. Themethod of claim 1, wherein the oxygen consumption in the substrateresulting from the metabolic process is observed.
 6. The method of claim1, wherein a redox reaction in the substrate resulting from themetabolic process is observed.
 7. The method of claim 1, whereby theproduction of CO₂ in the substrate resulting from the metabolic processis observed.
 8. The method of claim 1, whereby the electricalconductivity of the substrate resulting from the metabolic process isobserved.
 9. A method for the identification or determination of asubstance in a sample which comprises adding to the sample an entityselected from the group consisting of microorganisms and unicellularorganisms whose metabolism is influence by said substance, said entitybeing bound to an adsorbent having a specific binding power for saidentity, bringing the sample with the added entity into contact with anutrient-containing substrate to initiate metabolism of said entity,thereby to cause chemical or physical changes in the substrate, andobserving said changes.
 10. The method of claim 9, characterized in thata pH change, caused by the metabolism, is determined.
 11. A method claimin claim 9, characterized in that oxygen consumption, caused by themetabolism, is determined.
 12. The method of claim 9, characterized inthat redox status is determined for the reduction equivalents that areproduced by the metabolism.
 13. The method of claim 9, characterized inthat CO₂ produced by the metabolism is determined.
 14. The method ofclaim 9, characterized in that a change in electrical conductivity,caused by the metabolism, is determined.
 15. A method for theidentification or determination of an entity selected from the groupconsisting of microorganisms and unicellular organisms in a sample whichcomprises:(a) introducing the sample into a vessel containing anadsorbent with a specific binding power for said entity, to bind saidentity to said adsorbent; (b) removing unbound sample from said vessel;(c) introducing a fluid nutrient medium into said vessel; (d)maintaining said nutrient medium in contact with said adsorbent and theentity bound thereto to initiate metabolism of said entity; (e) removingthe nutrient medium from the vessel and observing the effects of saidmetabolism on said removed medium.